A five-chamber microbial desalination cell (MDC) with anode, cathode, one central desalination chamber and two concentrate chambers separated by ion exchange membranes was operated in batch mode for more than 60 days. The performance of the MDC was evaluated for chemical oxygen demand (COD) removal, total dissolved solids (TDS) removal and energy production. An average COD removal of 81 ± 2.1% was obtained using acetate-fed wastewater as substrate in the anodic chamber inoculated with mixed anaerobic sludge. TDS removals of 58, 70 and 78% were observed with salt concentration of 8, 20 and 30 g/L, respectively, in the middle desalination chamber. The MDC produced a maximum power output of 16.87 mW/m2 during polarization. The highest Coulombic efficiency of 12 ± 2.4% was observed in this system using mixed anaerobic sludge as inoculum. The system effectively demonstrated capability for simultaneous organic matter removal and desalination along with power generation.